Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility-stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this family, ATX also possesses lysophospholipase D (LPLD) activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to lysophosphatidic acid (LPA) and sphingosylphosphorylcholine to sphingosine-1-phosphate (S1P). The ATX products, LPA and S1P, are bioactive phospholipids associated with cell growth, motility, and survival, as well as with blood vessel formation and maturation. In a recently published study, we showed a potential positive feedback loop between VEGF, ATX expression, LPA, and VEGFR2 expression in ovarian cancer cell lines. Current work with human umbilical vein and human umbilical artery cells has shown that VEGF acting via VEGFR2 also stimulates ATX expression in endothelial cells. The resulting elevation in LPA levels modulates expression of Akt isomers, particularly Akt-2. In contrast, down-regulation of ATX secretion in human umbilical vein cells using antisense morpholino oligomers virtually abrogates cell motility responses to VEGF, LPA, ATX, and lysophosphatidylcholine and significantly reduced responses to S1P and sphingosylphosphorylcholine. Although ATX appears to affect both tumor angiogenesis and embryonic vascular development, the mechanisms for these effects are poorly understood. Our data suggest that the ATX role in angiogenesis is closely linked to VEGF expression and activity as well as to Akt regulation. Since the enzymatic activity of ATX is critical for its cancer-promoting activity, we have also continued to study the enzymatic active site. We have recently characterized two amino acids within the enzymatic active site in which point mutations result in partial loss of activity. Previously studied mutations are characterized by complete loss of phosphodiesterase and lysophospholipase D activity to all tested substrates. However, H226Q or H415Q mutant proteins have partial activities that varied with substrate, indicating that these two amino acids could be involved in ATX-substrate interactions.